Tris-HCl Buffer Research Articles

Investigation on the Effect of Fluorescence Quenching of Calf Thymus DNA by Piperine: Caspase Activation in the Human Breast Cancer Cell Line Studies.

In this study, we determined the interaction of piperine and calf thymus DNA (ct DNA) in Tris-HCl buffer solution at pH = 6.8 and also evaluated the binding mechanism through the data of multi-spectroscopic techniques along with thermal melting and viscosity measurements. The outcomes of fluorescence quenching confirmed the occurrence of interactions between piperine and ctDNA and pointed out the role of piperine as the quencher. In addition, the KSV values were measured at three different temperatures of 298, 303, and 308 K to be 4.5 × 107 M-1, 5.65 × 107 M-1, and 9.36 × 107 M-1, respectively, which suggested the dominance of dynamic mechanism as the fluorescence quenching of piperine-ctDNA. The thermodynamic parameters revealed the predominance of hydrophobic forces in the interaction of ctDNA with piperine. According to the resonance light scattering data, the formation of a complex between piperine and ctDNA led to the creation of a larger particle. Ethidium bromide (EB) and acridine orange (AO) displacement studies, along with the ionic effects of NaCl and KI assessments, confirmed the interaction of piperine-ctDNA through a groove binding mode. The melting temperature assay of ctDNA upon the addition of piperine concentration indicated the probable groove binding of piperine to ctDNA, which was affirmed by relative viscosity measurement as well. The lack of detecting any alterations in the circular dichroism (CD) spectrum of CD investigation verified as a characteristic sign of groove binding mechanism and also confirmed all the experimental results with regard to the binding of piperine-ctDNA complex. Next to observing a concentration and time-dependent cytotoxicity in MDA-MB-231 cells, the impact of piperine on increasing lipid peroxidation and decreasing the activity of superoxide dismutase was also noticed. Apparently, piperine is capable of inducing caspase-3 activity as well.

Solvent- and pH-Stable Eu(III)-Based Metal-Organic Framework with Phosphate-Ratio Fluorescence Sensing and Significant Proton Conduction.

Lanthanide-based metal-organic frameworks show good potential for applications due to their unique structures and functional properties. A highly thermally and acid-base stable Eu-MOF was synthesized by a solvothermal method with the molecular formula {[(CH3)2NH2]2[Eu2(NDDP)2(H2O)2]·H2O}n (Eu-MOF, H4NDDP = 5,5'-(naphthalene-2,6-diyl)diisophthalic acid). Eu-MOF takes a three-dimensional (4,4,8)-connected topology. The water molecules involved in the coordination, free water molecules, and [(CH3)2NH2]+ cations in the pore can be used as proton carriers. The proton conductivity attains 1.25 × 10-4 S cm-1 at room temperature and 2.42 × 10-3 S cm-1 at 70 °C and 98% relative humidity. Combined with the dual-emission properties from the ligands and Eu(III) ions enables Eu-MOF to be used as a ratiometric fluorescent sensor for phosphate efficiently and rapidly, with a limit of detection of 0.12 μM in the Tris-HCl buffer solution. These results provide a new approach for the construction of MOFs with high proton conductivity and a ratiometric fluorescence response.

Improved expression and purification of highly-active 3 chymotrypsin-like protease from SARS-CoV-2

Severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2) is the causative pathogen of coronavirus disease-19 (COVID-19). The COVID-19 pandemic has resulted in millions of deaths and widespread socio-economic damage worldwide. Therefore, numerous studies have been conducted to identify effective measures to control the spreading of the virus. Among various potential targets, the 3 chymotrypsin-like protease (3CLpro), also known as Mpro, stands out as the key protease of SARS-CoV-2, playing an essential role in virus replication and assembly, is the most prospective. In this study, we modified the commercial vector, pETM33-Nsp5-Mpro (plasmid # 156475, Addgene, USA), by inserting an autocleavage site (AVLQ) of 3CLpro and 6 × His-tag encoding sequences before and after the Nsp5-Mpro sequence, respectively. This modification enabled the expression of 3CLpro as an authentic N terminal protease (au3CLpro), which was purified to electrophoretic homogeneity by a single-step chromatography using two tandem Glutathione- and Ni-Sepharose columns. The enzyme au3CLpro demonstrated significantly higher activity (3169 RFU/min/μg protein) and catalytic efficiency (Kcat/Km of 0.007 μM−1.s-1) than that of the 3CLpro (com3CLpro) expressed from the commercial vector (pETM33-Nsp5-Mpro) with specific activity 889 RFU/min/μg and Kcat/Km of 0.0015 μM−1.s-1, respectively. Optimal conditions for au3CLpro activity included a 50 mM Tris-HCl buffer at pH 7, containing 150 mM NaCl and 0.1 mg/ml BSA at 37 °C.

Biochemical characterisation of a PL24 ulvan lyase from seaweed-associated Vibrio sp. FNV38

Ulvan is a green macroalgal cell wall polysaccharide that has tremendous potential for valorisation due to its unique composition of sulphated rhamnose, glucuronic acid, iduronic acid and xylose. Several potential applications such as production of biofuels, bioplastics and other value-added products necessitate the breakdown of the polysaccharide to oligomers or monomers. Research on ulvan saccharifying enzymes has been continually increasing over the last decade, with the increasing focus on valorisation of seaweed biomass for a biobased economy. Lyases are the first of several enzymes that are involved in saccharifying the polysaccharide and several ulvan lyases have been structurally and biochemically characterised to enable their effective use in the valorisation processes. This study investigates the whole genome of Vibrio sp. FNV38, an ulvan metabolising organism and biochemical characteristics of a PL24 ulvan lyase that it possesses. The genome of Vibrio sp. FNV38 has a diverse CAZy profile with several genes involved in the metabolism of ulvan, cellulose, agar, and alginate. The enzyme exhibits optimal activity at pH 8.5 in 100 mM Tris–HCl buffer and 30 °C. However, its thermal stability is poor with significant loss of activity after 2 h of incubation at temperatures above 25 °C. Breakdown product analysis reveals that the enzyme depolymerised the polysaccharide predominantly to disaccharides and tetrasaccharides.

A novel liquid crystal aptasensor via DNA aptamer conformational change for on-site detection of cocaine in sewage

A novel one-step liquid crystal (LC)-based aptasensor has been developed for on-site detection of trace substances in sewage analysis. The platform utilizes different mechanisms to simultaneously trigger the upper and bottom layers of LC orientation. By employing trace amounts of CTAB in Tris-HCl buffer, the upper layer of LC is aligned, while DNA aptamers modified on a single glass induce the orientation of the bottom layer. When targeting cocaine, the asymmetric single-stranded DNA aptamers transition to a symmetric Y-shape, causing the reorientation of LCs from planar to homeotropic. This results in an optical change from bright to dark in the LC image. The sensing procedure involves adding pre-buffered samples at pH 7.4 to the system in one step. The platform requires only 10 ng of DNA aptamers, which can be kept dry. It enables on-site detection of cocaine in sewage, exhibiting a linear calibration curve from 1 pM to 15 µM in water (R2 = 0.997) with a limit of detection of 0.5 pM. The aptasensor demonstrates quantitative recoveries ranging from 89.6% to 110.9% in sewage samples. This sensitive, robust, and portable LC-based aptasensor shows promise as a tool for on-site detection of trace substances.

Inhibitive determination of organophosphate pesticides using acetylcholinesterase and silver nanoparticle as colorimetric probe

Accurate and real-time detection of pesticides is needed to ensure food safety and ecosystem protection. A biosensor was developed to determine organophosphate pesticides using acetylcholinesterase (AChE) and silver nanoparticle (AgNP) immobilized on the alginate-chitosan film matrix. The sensing principle is based on organophosphate pesticides (OP) inhibiting AChE activity. AChE hydrolyzes acetylthiocoline (ATCh) to thiocholine (TCh) and acetic acid (AA). The -SH group on TCh induces AgNP aggregation, resulting in fading of the film's brownish-yellow color. The presence of OP in the sample inhibited AChE activity, leading to a decrease in TCh and color fading. The biosensor response is determined by measuring the Red Green Blue (RGB) value of the film. The biosensor offers rapid detection directly in-situ and real time without the use of sophisticated instruments, with optimal measurement conditions of pH 7, incubation time of 1 minute, and concentrations of AgNP, ATCh, and tris-HCl buffer of 10 μg/mL, 50, and 7 mM, respectively. Biosensors have excellent sensitivity, reproducibility, and selectivity. Measurement of OP levels in nine vegetable samples revealed that the proposed colorimetric probe's results agreed well with the GC result as the reference method. Therefore, this method can be applied in monitoring food and environment OP levels.

A highly active and stable chondroitin B lyase from Pedobacter schmidteae: Cloning, expression, and characterization

Chondroitin B lyase is a typical enzyme that catalyzes dermatan sulfate (DS) to produce oligosaccharides mainly 4,5-unsaturated dermatan sulfate disaccharides. It has been widely used in fields of medicine, functional food, and cosmetics because of its high selectivity. Here, a novel chondroitin B lyase from Pedobacter schmidteae (PsChonB) was cloned and heterologously expressed in Escherichia coli. The recombinant chondroitin B lyase had the highest activity in Tris-HCl buffer (100 mM, pH 8.0) at 40 °C. Characterization of the enzyme showed high stability of PsChonB, and its half-lives were 693 and 7949 min at 40 and 25 °C, and there was no significant decrease in activity after 30 days of storage at 4 °C. The specific activity, Km, Kcat, and Vmax of PsChonB were 395 U mg−1, 0.36 mg mL−1, 240.66 s−1, and 406.72 U mg−1. Molecular docking analysis of the enzyme indicated that the conserved residues Arg364, Arg363, His334, Arg318, Arg271, and Glu245 play vital roles in substrate binding. Compared with the previously reported chondroitin B lyases, PsChonB had the advantages of high activity and stability which indicated that it could be favorable for industrial production, clinic trials, and analytical the structures of glycosaminoglycans.

Additive manufacturing a novel bioceramic bone scaffold with MgO/Ca3(PO4)2: Microstructure, mechanical property and controllable degradation behavior

Biodegradable bone grafts have gained considerable attention in the field of clinical research as they can be used to avoid re-surgery to remove scaffolds. We fabricated biodegradable composite ceramic scaffolds using magnesium oxide and calcium phosphate following the vat photopolymerization technology. We studied the effects of different magnesium oxide content on the process of in vitro degradation of the composite scaffolds and discussed the degradation process by comparing the extent of degradation realized in Tris-HCl buffer and simulated body fluids. We also explored the variations in the mechanical properties of the scaffolds during degradation. The results revealed that the composite scaffolds maintained the strength standard for spongy bone (compressive strength σ: 2–20 MPa) even after 28 d of degradation. Additionally, the strength of the composite scaffold containing 80 wt% magnesium oxide was comparable to the strength standard recommended for cortical bones (σ: 100–200 MPa). Controllable degradation rates were recorded, and acceptable mechanical properties were observed for these scaffolds during degradation. The results reported herein can be potentially used to expand the application prospects of biodegradable scaffolds.

In vitro α-Amylase and α-Glucosidase Inhibitory Activity of Dioscorin from Dioscorea alata

Dioscorea alata is a species of yam found in many countries in Southeast Asia. It contains many bioactive compounds consisting of steroidal saponins, phenolic compounds flavonoids, polysaccharides, and proteins. Eighty to eighty-five of the soluble proteins in the tuber of D. alata are dioscorin. Dioscorin (31 kDa) is the major storage protein of yam tuber, exhibiting biochemical and immunomodulatory properties, such as antioxidant activity, reducing blood pressure and angiotensin I converting enzyme (ACE) inhibitors activity. The aims of this research focused on extraction of the total soluble protein from D. alata tuber, purification of dioscorin, and study of in vitro inhibitory activity of α-amylase and α-glucosidase of dioscorin extracts. The results showed that a maximum yield of total soluble protein was obtained from the buffers extraction using Tris-HCl buffer at pH 9.0 resulting in the protein concentration of 15.10 mg/g with a protein extraction yield of 31.56 ± 2.36%. The crude protein was further recovered using ammonium sulfate precipitation and dialysis. SDS-PAGE analysis showed that dioscorin from crude extracts was precipitated at the concentration of ammonium sulfate of 60% saturation. The purified dioscorin was obtained from anion exchange chromatography (DEAE-Sephacel) and its molecular weight of 31 kDa was estimated on SDS-PAGE. Furthermore, the α-amylase inhibitory activities of crude extract (1 mg/mL), purified dioscorin obtain from gastric digestion and purified dioscorin obtained from intestinal digestion were at 14.44% 2.21% and 78.13%, respectively. Moreover, the results showed that the α-glucosidase inhibitory activity of the crude extract, and purified dioscorin obtained from gastric digestion were at 10.56% and 2.61%, respectively.

Luminescent Diimine-Pt(IV) Complexes with Axial Phenyl Selenide Ligands

Luminescent diimine-Pt(IV) complexes [Pt(N^N)(Me)2(PhSe)2], (N^N = 2,2′-bipyridine (bpy, 1b), 1,10-phenanthroline (phen, 2b), and 4,4′-dimethyl-2,2′-bipyridine (Me2bpy, 3b), PhSe− = phenyl selenide were prepared and identified using multinuclear (1H, 13C{1H} and 77Se{1H}) NMR spectroscopy. The PhSe− ligands were introduced through oxidative addition of diphenyl diselenide to the non-luminescent Pt(II) precursors [Pt(N^N)(Me)2], N^N = (bpy, 1a), (phen, 2a), (Me2bpy, 3a), to give the luminescent Pt(IV) complexes 1b–3b. The UV-vis absorption spectra of 1b–3b are characterised by intense bands in the range 240–330 nm. We assigned them to transitions of essentially π−π* character with small metal and PhSe− ligand contributions with the help of TD-DFT (time-dependent density functional theory) calculations. The weak long-wavelength bands in the range 350–475 nm are of mixed ligand-to-metal charge transfer (L’MCT) (n(Se)→d(Pt)/intra-ligand charge transfer (IL’CT) (n(Se)→π*(Ph) or π(Ph)→π*(Ph))/ligand-to-ligand’ charge transfer (LL’CT) (L = N^N, L’ = PhSe−, M = Pt and n = lone pair) character. The Pt(IV) complexes showed broad emission bands in the solid state at 298 and 77 K, peaking at 560–595 nm with a blue shift upon cooling. Structured emission bands were obtained in the range 450–600 nm, with the maxima depending on the N^N ligands and the solvent polarity (CH2Cl2 vs. dimethyl sulfoxide (DMSO) and aqueous tris(hydroxymethyl)aminomethane hydrochloride (tris-HCl) buffer). The emissions originate from essentially ligand-centred triplet states (3LC) with mixed IL’CT/L’MCT contributions as concluded from the DFT calculation. Such dominating PhSe contributions to the emissive states are unprecedented in the world of luminescent diimine-Pt(IV) complexes.

Identification and characterisation of a major outer membrane protein from Methylacidiphilum fumariolicum SolV

The outer membrane (OM) protects Gram-negative bacteria against a hostile environment. The proteins embedded in the OM fulfil a number of tasks that are crucial to the bacterial cell. In this study, we identified and characterised a major outer membrane protein (WP_009059494) from Methylacidiphilum fumariolicum SolV. PRED-TMBB and AlphaFold2 predicted this protein to form a porin with a β-barrel structure consisting of ten antiparallel β-sheets and with a small amphipathic N-terminal α-helix in the periplasm. We purified soluble recombinant protein WP_009059494 from E. coli using Tris–HCl buffer with SDS. Antibodies were raised against two peptides in the two large extracellular loops of protein WP_009059494 and immunogold localisation showed this protein to be mainly present in the OM of strain SolV. In addition, this protein is tightly associated with the OM, and is resistant to extraction. Only a small amount can be isolated from the cell envelope using harsh conditions (SDS and boiling). Despite this resistance to extraction, WP_009059494 most likely is an outer membrane protein. A regular lattice could not be detected by negative staining TEM of strain SolV and isolated protein WP_009059494. Considering the specific ecological niche of strain SolV living in a geothermal environment with low pH and high temperatures, this major protein WP_009059494 may act as barrier to resist the extreme conditions found in its natural environment. In addition, we found an absence of the BamB, BamC and BamE proteins of the canonical BAM complex, in Methylacidiphilum and Methylacidimicrobium species. This suggests that these bacteria use a simple BAM complex for folding and transport of OM proteins.

Enzyme-Like Activity of Cerium Dioxide Colloidal Solutions Stabilized with L-Malic Acid

For the first time, stable aqueous colloidal solutions of cerium dioxide stabilized with L-malic acid have been obtained at ligand : CeO2 molar ratios of 0.2, 0.5, 1.0, and 2.0. Using dynamic light scattering, it has been shown that CeO2 sols are characterized by a narrow monomodal size distribution of aggregates, and the sols remain to be aggregatively stable in a Tris-HCl buffer solution. According to the chemiluminescence analysis of the enzyme-like activity of cerium dioxide sols with respect to hydrogen peroxide, the surface modification of the cerium dioxide particles with malic acid increases the enzyme-like activity of СеО2 up to 4.5 times.

Biochemical characterization of a new ulvan lyase and its applicability in utilization of ulvan and preparation of ulva oligosaccharides.

Ulva is globally distributed specie and has a high economic value. Ulvan is one of the main active substances in Ulva, which has a variety of biological properties. Ulvan lyase degrades ulvan through a β-elimination mechanism which cleaves the β-glycosidic bond between Rha3S and GlcA or IdoA. The complex monosaccharide composition of ulvan makes it promising for use in food and pharmaceutical applications. This thesis explores a putative ulvan lyase from Alteromonas sp. KUL_42. We expressed and purified the protein, performed a series of characterizations and signal peptide had been removed. The results showed that the protein molecular weight of ULA-2 was 53.97kDa, and it had the highest catalytic activity at 45°C and pH8.0 in Tris-HCl buffer. The Km and Vmax values were 2.24mg·mL-1 and 2.048μmol·min-1·mL-1, respectively. The activity of ULA-2 was able to maintain more than 80% at 20~30°C. ESI-MS analysis showed that the primary end-products were mainly disaccharides to tetrasaccharides. The study of ULA-2 enriches the ulvan lyase library, promotes the development and high-value utilization of Ulva resources, and facilitates further research applications of ulvan lyase in ulva oligosaccharides.

Ferguson plot analysis of multiple intermediate species of thermally unfolded bovine serum albumin

Ferguson plot was used to characterize the multiple intermediate species of bovine serum albumin (BSA) upon thermal unfolding. Differential scanning calorimetry showed an irreversible melting of BSA in Tris-HCl and phosphate buffers with a mid-transition temperature, Tm, of ∼68 °C. Thermally unfolded BSA was analyzed by agarose native gel electrophoresis stained by Coomassie blue and SYPRO Orange staining as a function of pH or protein concentration. SYPRO Orange was used to stain unfolded proteins. BSA heated at 70 and 80 °C, i.e., above the Tm, formed multiple intermediate species, which depended on the pH between 7.0 and 8.0, protein concentration and which buffer was used. These intermediate species were analyzed by Ferguson plot, which showed that BSA heated at 60 °C had a similar size to the native BSA, indicating that they are either native or native-like state consistent with no SYPRO Orange staining. The intermediate species observed at higher temperatures with the mobility less than that of the native BSA showed a steeper Ferguson plot and were stained by SYPRO Orange, indicating that these species had a larger hydrodynamic size than the native BSA and were unfolded.

Partial Purification and Characterization of the Acetylcholinesterase from Poecilia reticulata (Peters, 1859)

Poecilia reticulata (P. reticulata) is a species of tropical fish in the family Poeciliidae that is found all over the world and is a favorite in freshwater aquariums. The Malay name for this fish is "Ikan Gapi," although the English term "Guppy" has become more common. The Million Fish, or Rainbow Fish, as it's known in other parts of the world. The goals of this research were to char-acterize the acetylcholinesterase that will be partially purified from P. reticulata brain extract using ammonium sulphate fractionation followed by an affinity chromatography method. The result shows that ammonium sulphate fractionation gave better purification fold over the procainamide affinity chromatographic method with fold of purification of 3.38 compared to 0.64 for the latter. Polyacrylamide gel electrophoresis (SDS-PAGE) shows a dramatic reduction of protein bands compared to crude extract. A pH of 9 in a Tris-HCl buffer system and a temperature of 15 °C were found to be ideal for the activity of the enzyme. Based on its high Vmax and low Km with acetylthiocholine iodide (ATC) as a substrate compared to butyryl thiocholine iodide (BTC) and propionyl thiocholine iodide (PTC), the substrate specificity profile identified acetylcholinesterase (AChE) as the major enzyme present in the purified enzyme. The molecular weights of the en-zyme following affinity chromatographic purification are about 92 kDa.

Purification and characterization of human glycerol 3-phosphate dehydrogenases (mitochondrial and cytosolic) by NAD+/NADH redox method

Glycerol 3-phosphate (G3P) shuttle is composed of mGPDH and cGPDH and serves as the interface between carbohydrate- and lipid-metabolism. Recently, these metabolic enzymes have been implicated in type II diabetes mellitus but the detailed kinetic parameters and crystal structure of human mGPDH is unknown, though fewer studies on cGPDH are available. To characterize these enzymes, the human mGPDH and cGPDH genes were optimized and cloned into the pET-SUMO vector and pET-24a(+) vector, respectively, and over-expressed in Escherichia coli BL21 (DE3). However, SUMO-mGPDH was expressed as inclusion bodies. Hence, various culture parameters, solubilizing agents and expression vectors were used to solubilize the protein but they did not produce functional SUMO-mGPDH. Over-expression of SUMO-mGPDH along with molecular chaperone (pG-KJE8) produced a functional SUMO-mGPDH. The functional SUMO-mGPDH was purified and characterized using NAD+/NADH redox method. cGPDH was also over-expressed and purified for its characterization. DLS analysis and CD spectra of the purified proteins were performed. The mGPDH was a monomeric enzyme with MW of ∼74 kDa and displayed optimal activity in the Tris-HCl buffer (pH 7.4); while, cGPDH was a homodimer with a monomeric MW of ∼37 kDa and showed optimal activity in imidazole buffer (pH 8.0). The Kmapp was 0.475 mM for G3P, and 0.734 mM for DHAP. These methods may be used to characterize these enzymes to understand their role in metabolic disorders.

Effect of polydopamine coating on adhesion of resin composite to polyetherketoneketone (PEKK)

Statement of problemPolydopamine (PD) adsorbs on all kinds of surfaces by forming strong covalent bonds, and develops cohesive strength through self-polymerization, resulting on thin strong adhesive films. Thus, PD coating could be promising to improve the adhesion to the inert Polyetherketoneketone (PEKK). PurposeTo analyze different protocols of PD coating to improve gingiva veneering resin composite bonding to PEKK. Material and methodsMilled PEKK specimens were fabricated and ultrasonically cleaned. Dopamine hydrochloride (Sigma Aldrich) was mixed with TRIS-HCl buffer (Sigma Aldrich) at pH 8.5 to obtain concentration of 1, 2, and 4 mg/mL. Each concentration was applied for 7 and 14 h to coating PEKK surface. Controls groups without treatment, surface treatment (with and without air abrasion), and bonding treatment (PEKKBond (AnaxDent)) were used for comparisons (n = 36 each). Morphological topography was analyzed, and flowable gingiva colored composite resin (AnaxGum, Anaxdent) were bonded to PEKK surface. Specimens were aged for 24 h (37 °C) and the shear bond strength (SBS) was assessed in universal test machine (MPa). Statistical analysis was performed by using 2-way ANOVA followed by Tukey's tests (α = 0.05). ResultsAll main effects and interactions were significant (P < .05). In general, PD coating reduced the adhesion between PEKK and veneering composite (P < .001), regardless of the concentration or the time of application (P > .05). The conventional adhesive (PEKKBond) presented the highest SBS values, regardless prior to air abrasion treatment. ConclusionThe PD coating of PEKK does not improve the bond strength between gingiva veneering composite and PEKK. Clinical implicationsSurface conditioning of PEKK surface properties is of interest to the dental community and, therefore, many treatments have been suggested to increase the adhesiveness of such polymer. However, PD coating is not an efficient bonding agent for PEKK and should not be used in a clinical practice since it is time-consuming and reduces the adhesion to PEKK.

Biocatalysts based on complexes of carbon nanomaterials with cysteine proteases

The purpose of the research is to develop and study biocatalysts based on complexes of cysteine proteases with fullerenes and carbon nanotubes. During the formation of ficin complexes with fullerenes and carbon nanotubes, the activity of hybrid preparations was 70 and 45%, respectively. During the formation of papain complexes with fullerenes and carbon nanotubes, the proteolytic ability of the enzyme remained at the same level for the samples with fullerene and decreased by 27% for the preparations with carbon nanotubes. The formation of bromelain complexes with fullerenes and carbon nanotubes contributed to a decrease in the proteolytic activity of the biocatalyst by 18 and 48% as compared to the free enzyme. While determining the stability of complexes of nanomaterials and cysteine proteases during a 7-day incubation in 0.05 M tris-HCl buffer (pH 7.5) at 37 °C, we noticed a decrease in the proteolytic activity of the samples. Complexation with carbon nanoparticles and fullerenes increased the stability of ficin and bromelain, while the stability of papain in the complexes remained unchanged

Peroxidase activity as a biochemical marker of insecticide use in vegetables

The insecticides use is important for crop improvement and protection, but in excessive amounts, they would induce a dysfunction of metabolic enzymatic systems in plant tissues, leading to undesirable qualitative changes. In this context, we are interested in peroxidase (POD), an important enzyme in plant physiology but whose activity seems to be conditioned by the presence of insecticides in the soil. This work aims to study the impact of locally used insecticides (chlorpyrifos and dimethoate) on the activity of POD in parsley, onion, celery and garlic grown in soils treated or not. POD extraction was performed using Tris-HCl buffer (pH 7.3); its activity was measured using the substrate o-dianisidine in the presence of H2O2. Our result showed that POD activity for insecticide treated parsley, celery and onions increased by 30 % 127 % and 341 % respectively, however did not change significantly for garlic. Thus, the action of these chemicals is not trivial because they may alter non-target pathways, especially when doses are not adjusted accordingly. We found that insecticide stress would increase POD activity in all vegetables except garlic, which showed tolerance to insecticides. Our findings suggest that organic farming conditions could minimize peroxidase activity in parsley, celery and onion. We add that overproduction of POD negatively affects the quality and reduces the shelf life of vegetables, thus would be a very interesting biomarker of insecticide stress.

A centrifugal microfluidic platform for rapid detection of pathogens based on chitosan nucleic acid extraction and RAA-T7-CRISPR/Cas13a nucleic acid detection

This study reported a bacterial detection platform using chitosan electrostatic adsorption for nucleic acid purification, RAA-T7-CRISPR/Cas13a system for nucleic acid quantification and centrifugal microfluidic platform for full-automatic operation. First, the nucleic acids in the lysed bacterial sample were captured by chitosan modified in the microfluidic PDMS channel through electrostatic adsorption, followed by washing with sodium acetate buffer solution. Then, these captured nucleic acid templates were eluted using Tris-HCl buffer solution through restoring the electrical neutrality of chitosan. Finally, the eluted nucleic acid templates were transferred into the RAA-T7-CRISPR/Cas13a system and isothermally amplified at 37 °C, followed by real-time detection of fluorescent products. A centrifugal microfluidic chip was elaborately designed with a chitosan modified channel, eight functional chambers and three siphon valves to achieve sequential capture, washing, elution, amplification and detection of nucleic acids. This platform has the capacity of automatically detecting Salmonella within 1 h. The detection limit of this platform was 10 CFU/mL in chicken supernatant under the use of optimal conditions. The average recovery and standard deviation of this platform are 106.9% and 6.4%, respectively.